Glycopeptide compositions

ABSTRACT

The invention include glycopeptides having a glycoside and a peptide covalently bound through an amide bond. The glycopeptides may also include a diagnostic or therapeutic agent bound to the glycopeptide. A metal, such as a radionuclide, may also be chelated to the glycopeptide. Specific embodiments of the invention relate to glycopeptides made of chitosan covalently bound to a poly(amino acid) such as poly(glutamic acid) or poly(aspartic acid). Diagnostic agents conjugated to the glycopeptide may facilitate imaging. Specific therapeutic agents that may be conjugated to the glycopeptide include anticancer drugs, rheumatoid arthritis drugs, anticoagulants, anti-angiogenesis drugs, apoptosis drugs, osteoporosis drugs, steroids, and anti-inflammatory drugs. Some agents, such as radionuclides, may have both diagnostic and therapeutic effects. The glycopeptides may be made by combining a glycoside and a peptide in the presence of a carbodiimide and an acid group activator to form an amide bond between the glycoside and the peptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/071,975, filed on Mar. 4, 2005 and published as U.S. PatentApplication Publication No. 2006/0198786 on Sep. 7, 2006, the entiredisclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to glycopeptide compositions containing aglycoside and a peptide and their uses as biomaterials. Specificembodiments relate to chitosan-based glycopeptides. More specificembodiments relate to glycopeptides including chitosan and poly(aminoacids).

BACKGROUND

Biomaterials are constantly in demand for a variety of medical uses inbiological systems, such as humans and other mammals. These medical usesspan almost the entire breadth of medical treatments currently possible.For example, some biomaterials are used in medical devices or implantsboth for structural and biochemical purposes. Others are used as drugdelivery vehicles or even as drugs themselves.

The intended use often influences the type of material selected, but inmost uses a biodegradable or bioclearable material is preferred.Similarly, while some degree of toxicity or harmfulness may be toleratedor even preferred for certain biomaterials, in most applications it ispreferable that the material not be toxic.

Given the wide variety of uses and desired characteristics forbiomaterials, there is a constant demand for new biomaterials.

SUMMARY

The present invention relates to glycopeptides formed from at least oneglycoside covalently bound to at least one peptide. Although glycosidesand peptides, as well as agents conjugated thereto are often referred toin the singular throughout the specification and claims, given thepolymeric nature of the present invention, it will be readily understoodby one skilled in the art that glycopeptides may contain more than oneglycoside and more than one peptide. Further, because of multiplebinding sites, many agents of a given type may be bound to a singleglycopeptide. Methods to control the relative numbers of glycopeptidemonomers as well as the degree of substitution of any agents on polymersare well known in the art and may be applied to the present inventionthrough routine experimentation.

In one embodiment, the invention includes a glycopeptide having at leastone glycoside moiety covalently bound through an amide bond to at leastone peptide and additionally a diagnostic or therapeutic agentconjugated to one of either the glycoside or peptide moiety. Further,the glycopeptide may have a metal agent conjugated to the moiety nothaving a diagnostic or therapeutic agent.

The diagnostic or therapeutic agent may be conjugated to the glycosidethrough a carboxylic acid via a peptide bond linkage, in which case anymetal may be chelated to the peptide. Alternative, the diagnostic ortherapeutic agent may be conjugated to the peptide through an aminegroup via a peptide bond linkage, in which case any metal may bechelated to the glycoside. The glycopeptide may have a molecular weightof about 5,000 daltons to about 30,000 daltons.

The glycoside may include at least two covalently bound monomeric units.Further, the glycoside may include an aminated sugar. Specifically, theglycoside may be chitosan, collagen, chondroitin, hyauraniate, andheparin, and any combinations thereof. The glycoside may be selectedbased on ability to target endothelial cells such as vascular tissue,including extracellular proteins of vascular tissue, such as integrins.The glycoside may have a molecular weight of from about 3,000 daltons toabout 10,000 daltons.

The peptide may include a poly(amino acid) or a peptide or a giventargeting sequence. Poly(amino acids) having primarily acidic aminoacids may be preferred in some embodiments. The poly(amino acid) mayinclude poly(glutamic acid) and poly(aspartic acid), and anycombinations thereof. The peptide may make up about 5% to about 50% byweight of the glycopeptide. Further, the peptide may have a molecularweight of from about 750 daltons to about 3,000. The peptide may targetcancer cells.

The diagnostic or therapeutic agent may make up from about 10% to about60% by weight of the glycopeptide. Any diagnostic or therapeutic agentcapable to be conjugated to the glycopeptide may be used. In certainembodiments, the diagnostic agent may be an anticancer drug, rheumatoidarthritis drug, anticoagulant, anti-angiogenesis drug, apoptosis drug,osteoporosis drug, steroid, anti-inflammatory drug, or any combinationthereof. For example, the diagnostic or therapeutic agent may includemethotrexate or pamidronate.

In some embodiments, the glycopeptide may also include an additionalmetal diagnostic or therapeutic agent. Specifically, the metal may beconjugated via a chelating agent, such as DPTA. The metal may be aradionuclide, such as Tc-99m, Cu-60, Cu-61, Cu-62, Cu-67, In-111,T1-201, Ga-67, Ga-68, As-72, Re-186, Re-188, Ho-166, Y-90, Sm-153,Sr-89, Gd-157, Bi-212, and Bi-213, or any combinations thereof.Radionuclide metals may serve a dual purpose as both a diagnostic and atherapeutic.

Diagnostic agents may facilitate diagnosis in a variety of manners,including imaging. In some embodiments, the glycopeptides may be usedfor combined diagnosis and treatment and for dosage measurement in asubject. The may be accomplished by using a radionuclide, which bothfacilitates imaging and serves as an anti-cancer agent.

Alternatively, the glycopeptide may include a metal and a therapeuticagent, allowing one to measure the amount of the therapeutic deliveredto the target area in a subject. In yet another alternative, twodifferent glycopeptides may be used in combination. First, a diagnosticglycopeptide may be administered to a subject and the amount reachingthe target area measured. Next, the correct dosage for a therapeuticglycopeptide may be determined based on the assumption that it willreach the target area in amounts similar to the diagnostic glycopeptide.

Other embodiments of the present invention relate to a glycopeptideincluding a chitosan covalently bound to a poly(amino acid) via an amidebond. The chitosan may be an unmodified chitosan, or a modified version,such as alkyl sulfonated chitosan. Similarly, the poly(amino acid) maybe of any type, but in many embodiments it may be an acidic poly(aminoacid) such as poly(glutamic acid) or poly(aspartic acid).

Chitosan-based glycopeptides of the present invention may be used in thesame manner as glycopeptides of the present invention described above.For example, chitosan-based glycopeptide may also have a diagnostic ortherapeutic agent, including a metal, conjugated thereto.

Other embodiments of the present invention relate methods of makingglycopeptide, such as those described above. Specifically, a glycosideand a peptide may be combined in the presence of a carbodiimide and anacid group activator to form an amide bond between the glycoside andpeptide. The glycoside may be further reacted to form an amide or esterbond between the glycoside or peptide and a diagnostic or therapeuticagent. Additionally, a chelating agent may be conjugated to theglycoside or peptide to allow chelation of a metal.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete and thorough understanding of the present embodimentsand advantages thereof may be acquired by referring to the followingdescription taken in conjunction with the accompanying drawings. Thepatent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 illustrates glycopeptide compositions comprising glycosides andpeptides using chitosan and poly(glutamic acid) as examples in of fourtypes of arrangements depicted as FIG. 1A, FIG. 1B, FIG. 1C AND FIG. 1D.

FIG. 2 illustrates a synthesis reaction for formation of glycopeptide(FIG. 2A) and glycopeptide conjugates (FIG. 2B).

FIG. 3 illustrates two different glycopeptide conjugates having both ametal and another agent conjugated to the glycopeptide.

FIG. 4 illustrates structures of the glycopeptide and chitosan agentstested for tumor and bone localization ability.

FIG. 5 shows the cellular uptake of ^(99m)Tc-GP, ^(99m)Tc-PGA and^(99m)Tc-CH (100%) by mammary tumor cells in vitro.

FIG. 6 shows the results of assays for cellular uptake in breast cancercells (13762 cell line) in vivo. Planar scintography of ^(99m)Tc-CH-10(50%), ^(99m)Tc-CH-10 (100%), ^(99m)Tc-PGA (750) and ^(99m)Tc-G-P (1:2)in breast tumor cell line and inflammation-bearing rats (280-300mCi/rat, i.v. injection, acquired 500,000 count) was performed 120minutes after injection to compare tumor visualization. Tumor-to-Muscleand Tumor-to-inflammation ratios are shown. T=tumor, M=muscle andI=Inflammation.

FIG. 7 shows the results of assays for cellular uptake in bone in vivo.Planar scintography of ^(99m)Tc-Pamidronate (P), ^(99m)Tc-Glycopeptide(GP), ^(99m)Tc-Glycopeptide-Pamidronate (GPP) was performed 120 minutesafter injection. (300 μCi/rat, i.v. injection, acquired 500,000 count).

FIG. 8 also shows the results of assays for cellular uptake in bone invivo. A line profile demonstration of ^(99m)Tc-Pamidronate (P),^(99m)Tc-Glycopeptide (GP), ^(99m)Tc-Glycopeptide-Pamidronate (GPP) wasperformed 120 minutes after injection. (300 μCi/rat, i.v. injection,acquired 500,000 count).

DETAILED DESCRIPTION

The present invention relates to glycopeptide (GP) compositionscontaining a glycoside and a peptide and their uses as biomaterials. Insome embodiments, these biomaterials may be provided as a sterilizedpowder. Therapeutic or diagnostic agents may be conjugated to theglycopeptides.

In a specific embodiment, the glycoside and peptide may be joined by anamide bond. The glycoside may be an aminated sugar. The glycopeptide mayinclude between 5% to 50% peptide by weight. Too much peptide may resultin unacceptable levels of crosslinking between glycopeptides. Theglycoside may have a molecular weight of between about 3,000 to 10,000daltons. The peptide may have a molecular weight of between about 750 to3,000 daltons. The glycopeptide may have a molecular weight of betweenabout 5,000 to 30,000 daltons.

In a more specific embodiment, the glycopeptide may be made fromchitosan and a poly(amino acid), particularly poly(glutamic acid). Thisglycopeptide has a tumor targeting capacity without the need formodification to include specific targeting agents.

Chitosan (CH) is a polyaminosaccharide of particular interest in anumber of applications. Like many polyaminosaccharides, chitosan may bereadily harvested from naturally occurring materials. The primary sourceof chitosan is presently discarded shells of lobsters and crayfish orshrimp, although it may also be obtained from the shells of crabs andother crustaceans as well as from insect shells and fungi. Chitosan isnormally non-toxic and is compatible with a variety of living systems,including human tissues. However, like many other polyaminosaccharides,chitosan exhibits only limited solubility in water. To improvesolubility, alkyl sulfonated chitosan may be used. Alkyl sulfonatedchitosan is described, for example in U.S. patent application Ser. No.10/871,890, filed Jun. 18, 2004.

Other suitable glycosides include collagen, chondroitin, hyauraniate andheparin.

Poly(glutamic acid) (PGA) is also readily available commercially (SigmaChemical Company, St. Louis, Mo.) and may be synthesized in a variety ofmanners. PGA has a positive charge in physiological conditions and isbiodegradable, which may make it more compatible with biological uses.

Other peptides may be used in alternative embodiments of the invention.These peptides may include other poly(amino acids) as well as peptideshave a specific sequence or specific amino acid composition. In someembodiments, the peptide may serve a targeting function. In a specificembodiment, poly(aspartic acid) may be used. This likely enhances uptakeby tumor cells because they cannot manufacture aspartic acid internallyand much obtain it from an external source. For poly(amino acids)including amino acids having an acid group, the acid group may be usedfor later conjugation of the glycopeptide to various agents or it may beused for salt formation to improve solubility.

Glycopeptides of the present invention may have the glycoside andpeptide in a variety of arrangements. Four arrangements using chitosanand poly(glutamic acid) are show in FIG. 1. In various embodiments ofthe present invention, these different types of glycopeptides may beused as mixtures, or one or more structural arrangement may be separatedand used. Whether such separation of a particular structural arrangementis desirable may depend upon many factors, including the intended enduse and any conjugates to be added. In most synthesis methods, thestructure shown in FIG. 1A is likely to predominate.

One method of synthesizing a glycopeptide of the present invention isshow in FIG. 2. Alternative means of synthesis are possible. Forexample, the synthesis reaction may be designed to favor one type ofglycopeptide structure. In specific embodiments such as those shown inFIG. 2, the glycoside and peptide are conjugated using a carbodiimide asa coupling agent. Sulfo-NHS, in FIG. 2, serves as an acid groupactivator, facilitating glycopeptide formation. Other acid groupactivators may also be used to form glycopeptides of the presentinvention.

While the glycopeptides of the present invention may exhibit usefulbiological properties on their own, a large variety of agents may alsobe conjugated to the glycopeptides. Relevant agents include targeting,imaging and therapeutic agents. Multiple agents or types of agents maybe conjugated to the same glycopeptide molecule at the same time. Inspecific embodiments, the agent may comprise 10% to 60% by weight of theglycopeptide conjugate.

Although the glycopeptide inherently targets tumor tissue, agents tofurther increase tumor targeting or to make it more specific may beconjugated. Agents to target other tissue, such as pamidronate to targetbone, may also be conjugated. Methotrexate may be used to target folatereceptors. Many imagining agents include metals that may be provided byfirst conjugating a chelating agent, such as DPTA. These may be used tochelate valent metal ions such as ^(99m)Tc, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁷Cu,¹¹¹In, ²⁰¹Tl, ⁶⁷Ga, ⁶⁸Ga, ⁷²As, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁶⁶Ho, ⁹⁰Y, ₁₅₃Sm, ⁸⁹Sr,¹⁵⁷Gd, ²¹²Bi, and ²¹³Bi. These metal chelates may be used to imagedisease lesions. In some embodiments, the carboxyl and amino groups ofthe glycopeptide itself may be sufficient to chelate metal ions.

Therapeutic agents are most likely to be specific for the targetedtissue, such as chemotherapeutics and other anti-cancer drugs whentumors are targeted. Rheumatoid arthritis, anticoagulant,anti-angiogenesis, apoptosis, steroid, anti-inflammatory, andosteoporosis therapeutics may also be conjugated.

Therapeutic agents may be conjugated in any acceptable manner, butbiolabile bonds, such as amide or ester bonds, may be used in manyembodiments. FIG. 2B illustrates one method of conjugating anosteoporosis therapeutics, pamidronate. The same or similar methods maybe used to conjugate other agents. Agents may be conjugated to theglycopeptide using either a carboxylic acid or an amine group on theagent. Particularly when water-insoluble agents are conjugated, DMF orDMSO may be added to the reaction.

In specific embodiments, both imaging and therapeutic conjugates may beprovided to a subject. The imaging complex may then be used to determinethe optimal or recommended dosage of unlabeled therapeutic complex basedon how much of the imaging complex localizes to the target areas.

Some imaging complexes may use small metal ions that also haveradiotherapeutic effects. These complexes may be imaged to directlydetermine internal radiotherapeutic dosages in a subject.

Similarly, if radiochemicals or other imaging agents are incorporated inthe same complex as a therapeutic agent, dosage of the therapeutic agentin the subject may be directly measured. Examples of such complexes areshown in FIG. 3.

Conjugation of agents to the glycopeptide may provide for sustainedrelease of the agents, particularly in a biological system, such as amammal. Conjugation may also increase the effective water-solubility andtherapeutic index of agents that are poorly water soluble.

Tumor-Related Applications

Embodiments of the present invention may be used to treat tumors,particularly through delivery of cytotoxic agents. Delivery of cytotoxicagents, as opposed to merely cytostatic agents, has often provedproblematic in previous treatments. Although the glycopeptides of thepresent invention may be used to deliver cytostatic agents, the abilityof many of them to deliver cytotoxic agents as well increase their valueas a therapy vehicle.

In specific embodiments, the glycopeptide used contains chitosan andeither poly(glutamic acid) or poly(aspartic acid). These embodimentstarget tumor tissues, most likely through angiogenesis, which occurs ata vastly increased rate in tumor tissue. Vascular cells, andparticularly the integrin molecules located on them, are targeted bypolysaccharides (e.g. collagen, chondroitin, hyauraniate, chitosan).This vascular targeting helps prevent drug resistance of tumor cellsbecause it does not target tumor tissue directly. Additionally, tumortissue exhibits an increased need for amino acids and most cells havesurface receptors for certain amino acids, such as glutamic acid andaspartic acid, allowing the poly(amino acid) portion to serve atargeting function as well. Specifically, the poly(amino acid) is mostlikely taken up by the tumor cells.

The tumor targeting capacity of glycopeptides of the present inventionhas been shown with gamma imaging using a ^(99m)Tc-labeledchitosan/poly(glutamic acid) glycopeptide. ^(99m)Tc-labeledchitosan/poly(glutamic acid) glycopeptide may be used to quantify thedose needed fro treatment. Ultimately, ^(99m)Tc-labeledchitosan/poly(glutamic acid) glycopeptide may predict patients who mayrespond to therapy and be used in their selection. ¹⁸⁸Re may also beused as a radiotherapeutic to treat many tumors. ¹⁸⁸Re is most effectiveif it remains with the glycoside whether in the vasculature orinternalized into a tumor. ¹⁸⁸Re is a beta and gamma (15%) emitter andhas a half life of 17 hours. The tissue penetration is 5-7 mm, which canbe used to both image and treat large tumors at the same time.

The targeting capacity assists in the delivery of chemotherapeutics withpoor water solubility and can thus increase the therapeutic index(toxicity/efficacy) of such agents. Additionally, because thetherapeutics are gradually released from the glycopeptide, this alsocontributes to the therapeutic index and helps lessen acute systemictoxicity.

Bone-related Applications

In specific embodiments, a pamidronate may be conjugated to aglycopeptide of the present invention. This method of conjugation isshown in FIG. 2. Pamidronate is an osteoclast agent. Insignificanttoxicity was observed using chitosan/poly(glutamic acid) withpamidronate conjugated. Pamidronate exhibited quick renal clearance.Imagining studies have shown that this composition targets bone. Thesecompositions may be used to treat bone degeneration diseases, such asosteoporosis.

The following examples are provided to further describe selectedembodiments of the present invention.

EXAMPLES Example 1 Synthesis of Glycopeptide

During hydrolysis of chitosan, various molecular weights and percentagesof amino group conversions were prepared. Molecular weight andpercentage amino group conversions are noted herein as “CH[molecularweight] [amino conversion]%”. For example, CH10 designates chitosan witha molecular weight of 10,000 with 100% hydrolysis of the acetamide groupto form an amino group.

In a typical synthesis, to a stirred solution of chitosan (CH10, 100%),(200 mg, MW. 10,000-20,000) in water (5 ml), sulfo-NHS (232.8 mg, 1.07mmol) and 3-ethylcarbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (EDC) (204.5 mg, 1.07 mmol) (Pierce Chemical Company,Rockford, Ill.) were added. Poly(glutamic acid) (400 mg, MW. 750-3,000)was then added. The mixture was stirred at room temperature for 24hours. The mixture was dialyzed for 48 hours using Spectra/POR molecularporous membrane with cut-off at 10,000 (Spectrum Medical IndustriesInc., Houston, Tex.). After dialysis, the product was filtered andfrozen dried using lyophilizer (Labconco, Kansas City, Mo.). Theglycopeptide in the salt form weighed 568.8 mg. The compositions of fourtypes of resulting glycopeptides are shown in FIG. 1. The glycopeptidemixture was used in the remainder of these examples, although isolationand independent use of, for example, each of the four types ofglycopeptides, is possible.

Example 2 Radio Labeling of Glycopeptide with ^(99m)Tc

Glycopeptide (5 mg) was dissolved in 0.2 ml of water and tin chloride(0.1 mg in 0.1 ml of water) was added at room temperature. Sodiumpertechnetate (5 mCi) was added. Radiochemical purity was determined byTLC (ITLC SG, Gelman Sciences, Ann Arbor, Mich.) eluted withMethanol:Ammonium acetate (1:4). From radio-TLC (Bioscan, Washington,D.C.) analysis, the radiochemical purity was more than 95%.

Example 3 Synthesis of Glycopeptide-Pamidronate Conjugates

Pamidronate (100 mg, 0.24 mmol) was dissolved in 1 ml of sodiumbicarbonate (1N), sulfo-NHS (91.8 mg, 0.43 mmol) and 3-ethylcarbodiimide1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-HCl (EDC) (81.2 mg,0.43mmol). A solution of glycopeptide (200 mg) in 5 ml of deionizedwater was added. The solution was left stirring for 24 hr at roomtemperature. After dialysis (MW:10,000) and lyophilization, the yield ofpamidronate-glycopeptide was 250 mg. The synthesis scheme is shown inFIG. 2.

Example 4 In vitro Cell Culture Assay

To evaluate whether glycopeptides have tumor targeting potential,mammary tumor cell line (13762) was selected. The cells were plated to12 well tissue culture plates at a density of 50,000 cells per well. Thecells were incubated with 4 μCi (0.148 MBq) of ^(99m)Tc-labeledglycopeptide (GP), poly(glutamic acid) (PGA), chitosan (CH10, 50%), orchitosan (CH10) (100 μg/well). The structures of the agents tested areshown in FIG. 4. Cells were incubated with radiotracers at 37° C. at0.5-2 hrs. After incubation, cells were washed with ice-coldphosphate-buffered saline (PBS) twice and trypsinized with 0.5 ml oftrypsin solution. Then cells were collected and the radioactivity wasmeasured by gamma counter. Data are expressed in mean±SD percent uptakeratio of three measurements. There was similar cellular uptake betweenglycopeptide and PGA (FIG. 5). However, glycopeptide is preferred inactual biological systems because it targets both vascular tissue andcellular receptors, unlike PGA with targets cells alone.

Example 5 Tumor Scintographic Imaging Studies

To demonstrate whether glycopeptide could specifically target tumortissue, a group of female Fischer 344 tumor-bearing (right leg) ratswith or without turpentine-induced inflammation (left leg) wereadministered with 300 μCi of ^(99m)Tc-labeled glycopeptide, chitosan(50% and 100%), or poly(glutamic acid) (PGA). Scintographic images,using a gamma camera equipped with low-energy, parallel-hole collimator,were obtained at 0.5, 2 and 4 hrs. The tumor could be visualized well atall times. Tumor-to-muscle and tumor-to-inflammation ratios inglycopeptide group as compared to peptide and chitosan groups werehigher at 0.5-3 hrs. Selected images are shown in FIG. 6.

Cellular uptake assays indicated that glycopeptide and glutamate peptidehad higher uptake (0.4-0.5%) than chitosan (0.2%). Biodistribution of^(99m)Tc-glycopeptide in breast tumor-bearing rats showed increasedtumor-to-tissue count density ratios as a function of time. Planarimages confirmed that the tumors could be visualized clearly. At 2 hrs,tumor/muscle ratios for glycopeptide, glutamate peptide and chitosanwere 3.9, 3.0 and 4.89. Although tumor/muscle rations are higher forchitosan alone, use of the glycopeptide is preferred because it targetsboth cells and vasculature. Additionally, glycopeptide exhibits bettertissue retention overall.

Example 6 Tumor Response to Paclitaxil Treatment

To assess anti-angiogenic treatment response, rats were treated withpaclitaxel (40 mg/kg, iv), followed by imaging with^(99m)Tc-glycopeptide on day 4. Tumor uptake and in situ hybridization(ISH) and TUNEL assays were conducted pre- and post-paclitaxeltreatment.

In rats treated with paclitaxel, no marked tumor progression wasobserved compared to ^(99m)Tc-glycopeptide baseline on day 4. Tumornecrosis was clearly seen post-treatment.

There was a correlation between tumor uptake and cellular targetsexpression as demonstrated by ISH and TUNEL assays.

Example 7 Bone Scintographic Imaging Studies

To demonstrate glycopeptide could be used to target bone, normal femaleFischer 344 rats (125-175g) were administered with 300 μCi of^(99m)Tc-labeled pamidronate, the glycopeptide andglycopeptide-pamidronate conjugate. Glycopeptide-pamidronate was able totarget bone (FIGS. 7 and 8).

Example 8 Bone Loss Prevention

Glycopeptide-pamidronate conjugate, glycopeptide, or pamidronate will beadministered in various dosages to female rate whose ovaries havepreviously been removed. Oovarectomy is strongly correlated withosteoporosis-like bone loss in rats. This bone loss may be observed overa period of several months. Because glycopeptide-pamidronate conjugateexhibits bone-targeting tendencies, it is expected that itsadministration will lessen or prevent oovarectomy-associated bone lossin female rats. Further, because pamidronate targets bone poorly,improved results are expected when using the glycopeptide conjugate asopposed to pamidronate alone.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalternations can be made herein without departing from the spirit andscope of the invention as defined by the following claims.

1. A glycopeptide comprising: a glycoside moiety; a peptide moiety; anda diagnostic or therapeutic agent, wherein the glycoside moiety iscovalently bonded via an amide bond to the peptide moiety and thediagnostic or therapeutic agent is conjugated to the peptide moiety. 2.The glycopeptide of claim 1, further comprising a metal conjugated tothe glycoside moiety.
 3. The glycopeptide of claim 2, wherein thediagnostic or therapeutic agent is conjugated to the peptide moietythrough an amino group via a peptide bond linkage and the metal ischelated to the glycoside.
 4. The glycopeptide of claim 1, wherein theglycoside moiety comprises at least two covalently bound monomeric unitsand wherein the peptide moiety comprises a poly(amino acid).
 5. Theglycopeptide of claim 1, wherein the glycoside moiety comprises anaminated sugar.
 6. The glycopeptide of claim 1, wherein the glycosidemoiety comprises chitosan and the peptide moiety comprises poly(glutamicacid) or poly(aspartic acid).
 7. The glycopeptide of claim 1, whereinthe glycopeptide comprises the peptide moiety in an amount of from about5% to about 50% by weight of the glycopeptide.
 8. The glycopeptide ofclaim 1, wherein the glycoside moiety has a molecular weight of fromabout 3,000 daltons to about 10,000 daltons, and wherein the peptidemoiety has a molecular weight of from about 750 daltons to about 3,000.9. The glycopeptide of claim 1, wherein the glycopeptide has a molecularweight of from about 5,000 daltons to about 30,000 daltons.
 10. Theglycopeptide of claim 1, wherein the glycoside is selected from thegroup consisting of: collagen, chondroitin, hyauraniate, and heparin,and any combinations thereof, and wherein the peptide is selected fromthe group consisting of: poly(glutamic acid) and poly(aspartic acid),and any combinations thereof.
 11. The glycopeptide of claim 1, whereinthe diagnostic or therapeutic agent is selected from the groupconsisting of: anticancer drugs, rheumatoid arthritis drugs,anticoagulants, anti-angiogenesis drugs, apoptosis drugs, osteoporosisdrugs, steroids, anti-inflammatory drugs, and any combinations thereof.12. The glycopeptide of claim 1, wherein the diagnostic or therapeuticagent comprises methotrexate or pamidronate.
 13. The glycopeptide ofclaim 2, wherein the metal comprises a radionuclide.
 14. Theglycopeptide of claim 13, wherein the metal is selected from the groupconsisting of: Tc-99m, Cu-60, Cu-61, Cu-62, Cu-67, In-111, T1-201,Ga-67, Ga-68, As-72, Re-186, Re-188, Ho-166, Y-90, Sm-153, Sr-89,Gd-157, Bi-212, and Bi-213, and any combinations thereof.
 15. Theglycopeptide of claim 1, wherein the glycopeptide comprises thediagnostic or therapeutic agent in an amount of from about 10% to about60% by weight of the glycopeptide.
 16. The glycopeptide of claim 1,wherein the glycoside targets vascular endothelial cells and the peptidetargets cancer cells.
 17. A glycopeptide comprising: chitosan; and apoly(amino acid), wherein the chitosan and poly(amino acid) arecovalently bound through an amide bond.
 18. The glycopeptide of claim17, wherein the chitosan comprises alkyl sulfonated chitosan.
 19. Theglycopeptide of claim 17, wherein the poly(amino acid) comprisespoly(glutamic acid) or poly(aspartic acid).
 20. The glycopeptide ofclaim 17, further comprising a diagnostic or therapeutic agentconjugated to the poly(amino acid).
 21. The glycopeptide of claim 17,further comprising a metal conjugated to the chitosan.
 22. A method ofproducing a glycopeptide comprising combining a glycoside and a peptidein the presence of a carbodiimide and an acid group activator to form anamide bond between the glycoside and peptide.
 23. The method of claim22, further comprising forming an amide or ester bond between thepeptide and a diagnostic or therapeutic agent.
 24. The method of claim22, further comprising conjugating a chelating agent and a metal to theglycoside.